Compositions for enhancing delivery of nucleic acids to cells

ABSTRACT

This invention provides methods and compositions for enhancing transfer of an agent into a cell. The agents can include polypeptides, polynucleotides such as genes and antisense nucleic acids, and other molecules. In some embodiments, the agents are modulating agents that can modulate a cellular activity or function when introduced into the cell. The methods and compositions are useful for introducing agents into individual cells, as well as cells that are present as a tissue or organ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/889,355,filed Jul. 8, 1997, which is a continuation-in-part of U.S. Ser. No.08/584,077, filed Jan. 8, 1996, now U.S. Pat. No. 5,789,244 each ofwhich is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

This invention pertains to the field of delivering therapeutic and otheragents to cells. Genes, polypeptides, and other molecules are among theagents that can be delivered using the compounds and methods of theinvention. The cells can be present individually or as a biologicaltissue or organ.

Delivery of a compound into a cell is a first critical step for manydiagnostic and therapeutic processes. Gene therapy, for example, is ahighly promising tool for therapeutic and other uses that requiresdelivery of a nucleic acid to a cell. For example, distinct approacheshave been developed to treat neoplasms based on gene transfer methods.Methods have been developed to correct specific lesions at definedgenetic loci which give rise to neoplastic transformation andprogression (Spandidos et al., Anticancer Res. 10:1543-1554 (1990);Banerjee et al., Cancer Res. 52:6297-6304 (1992)). Overexpression ofdominant oncogenes may be addressed using techniques to inhibit thetransforming gene or gene product. Loss of tumor suppressor genefunction may be approached using methods to reconstitute wild-type tumorsuppressor gene function (Goodrich et al., Cancer Res. 52:1968-1973(1992)). Besides these methods to achieve mutation compensation, genetictechniques have been developed to specifically and selectively eradicatetumor cells. These approaches of molecular chemotherapy rely on specificexpression of toxin genes in neoplastic cells (Abe et al., Proc Soc ExpBiol Med. 203:354-359 (1993)). Finally, gene transfer methods have beenused to achieve antitumor immunization. These methods of geneticimmunopotentiation use techniques of genetic immunoregulation to enhanceimmune recognition of tumors. Consequently, a variety of distinctapproaches have been developed to accomplish gene therapy of cancer.

A high incidence of mutations has been observed in tumor suppressorgenes, such as p53 and RB, in the case of carcinoma of the bladder(Fujimoto et al., Cancer Res. 52:1393-1398 (1992); Cairns et al.,Oncogene 6:2305-2309 (1991)). For such genetic lesions of tumorsuppressor genes, reversion of the neoplastic phenotype can bedemonstrated with replacement of the corresponding wild-type tumorsuppressor gene (Spandidos, Id.; Banerjee, Id.).

Carcinoma of the bladder represents a significant source of morbidityand mortality. Bladder cancer ranks 10th in males and 12th in females incancer related mortality (Cancer Facts and Figures, Amer. Can. Soc. 5:11(1995)). Therapies available for the treatment of bladder cancer includeadjuvant chemotherapy or immunotherapy, transurethral resection ofsuperficial disease, radical cystectomy or radiotherapy which is oftencombined with systemic chemotherapy. Despite these therapeutic options,overall survival has not changed appreciably. (Id.) Thus, newtherapeutic modalities must be developed for the treatment of bladdercancer.

Gene therapy strategies have been developed as an alternativetherapeutic approach (See for example, Brewster et al., Eur Urol25:177-182 (1994); Takahashi et al., Proc Natl Acad Sci USA 88:5257-5261 (1991); Rosenberg, S A, J. Clin Oncol. 10:180-199 (1992)).Successful treatment of cancer and other conditions in a human or otheranimal can depend upon an adequate amount of a therapeutic agententering the cells, and upon a large enough proportion of target cellstaking up the therapeutic agent.

Many other therapeutics and other modulating agents are polypeptides or,for example, small molecules. Again, the amount of the agent thatreaches a target cell population can have a great impact on the efficacyof treatment. Therefore, a need exists for compounds and methods thatcan enhance the amount of an agent that is delivered to a cell or apopulation of cells.

The present invention fulfils this and other needs.

SUMMARY OF THE INVENTION

The invention provides compounds that can enhance delivery of an agentto cells. The delivery enhancing compounds of the invention typicallyhave a Formula I:

wherein:

m and n are the same or different and each is an integer from 2-8; R isa cationic group or

X₁ is selected from the group consisting of:

and X₂ and X₃ are each independently selected from the group consistingof a saccharide group,

wherein at least one of X₂ and X₃ is a saccharide group when R is

Some examples of preferred delivery enhancing compounds of the inventionare those that have a Formula III, IV, or V as shown in FIG. 21.

In some embodiments, the delivery enhancing compounds have a Formula II:

wherein X₁ and X₂ are selected from the group consisting of

and X₃ is a saccharide group.

Also provided by the invention are methods of delivering an agent tocells by administering the agent to the cells in a formulation thatincludes a delivery enhancing compound of Formula I.

In additional embodiments, the invention provides compositions fordelivering an agent to cells. The compositions include the agent to bedelivered and a delivery enhancing compound of Formula I.

A further aspect of the invention is a method of treating cancer,including bladder cancer, by administering to a cell a therapeuticallyeffective amount of a therapeutic agent that is formulated in a buffercomprising a compound of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the influence of formulation on adenovirus mediated genetransfer and expression in the rat bladder epithelium after intravesicaladministration.

FIG. 2 depicts adenovirus transgene expression in bladder epithelialcells after intravesical administration.

FIG. 3 depicts dose dependent adenovirus transgene expression in the ratbladder after intravesical administration.

FIG. 4 depicts a reverse-transcriptase polymerase chain reaction(RT-PCR) analysis of recombinant adenovirus transgene expression in themouse bladder after intravesical administration.

FIG. 5 depicts a time course of recombinant adenovirus transgeneexpression in bladder, kidney, and liver tissue after intravesicaladministration of the virus.

FIG. 6 depicts recombinant adenovirus transgene DNA in bladder andkidney homogenates after intravesical administration.

FIG. 7 depicts improvement of gene transfer to bladder epithelium usinga Big CHAP (N,N,bis-(3-D-gluconamidopropyl)-cholamide (CALBIOCHEM®Biochemicals, San Diego, Calif.) formulation.

FIG. 8 depicts improvement of gene transfer to bladder epithelium usingdifferent concentrations of recombinant adenovirus in a 7 mM Big CHAPformulation.

FIG. 9 depicts enhancement of recombinant adenovirus transgeneexpression in bladder tissue by using an ethanol (ETOH) or Big CHAPformulation.

FIG. 10 depicts gene transfer to tumors using a 4 mM Big CHAPformulation.

FIG. 11 depicts transgene transfer to pig bladder epithelium.

FIG. 12 depicts the expression of p53 in tumor tissue.

FIG. 13 depicts gene transfer to the mucosa of rat ileum.

FIG. 14 is a photograph of bladder sections from rats, wherein theability of Big CHAP from two sources to enhance gene transfer wascompared. The more intense Xgal staining in the lower row in comparisonto the upper row demonstrated a greater enhancement of gene transfer byBig CHAP from CALBIOCHEM® in comparison to Big Chap from Sigma (SigmaChemical Company, St. Louis, Mo.).

FIG. 15A and B depict thin layer chromatography (TLC) of Big CHAP fromCALBIOCHEM® and Sigma. Only one distinct band developed from the sampleof BC-Sigma (FIG. 15B), while three additional bands became evident inthe sample of BC-CALBIOCHEM® (FIG. 15A).

FIG. 16 depicts TLC of Big CHAP impurities. The lanes are labeled asfollows: Lane 1: Big CHAP (CALBIOCHEM®); Lane 2: Impurity I; Lane 3:Impurity II; Lane 4: Mixture of Impurity II and III; Lane 5: ImpurityIII; Lane 6: Big CHAP (CALBIOCHEM®) pure; Lane 7: Big CHAP(CALBIOCHEM®).

FIG. 17 is a photograph of bladder sections from rats, wherein theability of increasing concentrations of Big CHAP (Sigma) to enhance genetransfer was compared to a Big CHAP (CALBIOCHEM®) standard. The moreintense Xgal staining indicated enhanced gene transfer at higherconcentrations of Big CHAP (Sigma).

FIG. 18 is a photograph of bladder sections from rats, wherein theability of Big CHAP (CALBIOCHEM®) and Big CHAP (Sigma) afterpurification to enhance gene transfer was evaluated and compared tonon-purified Big CHAP from those sources as a control. The intensity ofthe Xgal staining indicated a reduced ability to enhance gene transferafter Big CHAP from either source had been purified by columnchromatography.

FIG. 19 is a photograph of bladder sections from rats, wherein theability of Big CHAP (CALBIOCHEM®) and Big CHAP (Sigma) afterpurification to enhance gene transfer was evaluated and compared tonon-purified Big CHAP from those sources and to Impurities I and acombination of impurity II and impurity III. The intensity of the Xgalstaining demonstrated an enhancement of gene transfer with 6 mg/ml ofthe combination of Impurity II and Impurity III.

FIG. 20 is a photograph of bladder sections from rats, wherein theability of Big CHAP (Sigma) after purification to enhance gene transferwas evaluated and compared to purified Big CHAP (Sigma) reconstitutedwith Impurity II, Impurity III, or a synthetic analog of Impurity II.The intensity of the Xgal staining demonstrated an enhancement of genetransfer when the purified Big CHAP (Sigma) was reconstituted. Big CHAP(CALBIOCHEM®) is included as a control.

FIG. 21 shows the structures of Syn3 and two water-soluble analogs ofSyn3. The domain of Syn3 that is conserved in the two analogs isindicated as “A”. The analogs A-TMA and A-HCl resulted from thesubstitution of trimethylammonium chloride (A-TMA) or hydrochloride(A-HCl) for the lactose moiety of Syn3.

FIG. 22 shows the structure, MALDI-MS, and ¹H-NMR of Impurity 1.

FIG. 23 shows the structure, MALDI-MS, and ¹H-NMR of Impurity 2.

FIG. 24 shows the structure, MALDI-MS, and ¹H-NMR of Impurity 3.

FIG. 25 shows a pathway for the synthesis of Impurity 2.

FIG. 26 shows a pathway for the synthesis of Syn3. An alternativepathway for Syn3 synthesis is shown in FIG. 34.

FIG. 27A-FIG. 27C demonstrate that I3A (Syn3) enhancesadenovirus-mediated β-galactoside expression. High levels of genetransfer were obtained when using I3A at 0.5 mg/ml in 7.8 mM Big CHAP(FIG. 27A). Controls are shown for comparison: no I3A (FIG. 27B) and asthe positive control, 7.8 mM Big CHAP Calbiochem Lot #679693 (FIG. 27C).

FIG. 28A and FIG. 28B show the results of a titration of gene transferenhancing activity of I3A (Syn3). Reduction of I3A to 0.25 mg/ml in 3.9mM Big CHAP (FIG. 28A) still yielded high levels of gene transferactivity compared to the gene transfer activity obtained when using I3Aat 0.5 mg/ml in 7.8 mM Big CHAP (FIG. 28B). At time of fixation,bladders treated with 0.25 mg/ml I3A appeared to have less inflammationthan those treated with 0.5 mg/ml I3A.

FIG. 29A-FIG. 29B show a comparison of I3A and Syn3 gene transferactivity. High levels of β-galactosidase activity were obtained usingI3A at 1 mg/ml in 0.1% Tween 80 (FIG. 29A). Approximately equal levelsof gene transfer were obtained using Syn3 at 1 mg/ml in 0.1% Tween 80(FIG. 29B).

FIG. 30A-FIG. 30D shows a comparison of Syn3 gene transfer activity in0.1% Tween 80 vs. 7.8 mM Big CHAP. Using Syn3 at 1 mg/ml in 0.1% Tween80 (FIG. 30A) resulted in levels of gene transfer that were comparableto those obtained when using Syn3 at 0.5 mg/ml in 7.8 mM Big CHAP (FIG.30C). Shown also are negative controls (no Syn3) when using either 0.1%Tween 80 (FIG. 30B) or 7.8 mM Big CHAP (FIG. 30D).

FIG. 31A-FIG. 31D show a comparison of Syn3 gene transfer activity atequal concentrations in Big CHAP and Tween 80 detergents. When Syn3 wasdissoluted at 0.5 mg/ml in 7.8 mM Big CHAP (FIG. 31A), very high levelsof gene transfer were obtained. In comparison, the gene transferactivity of Syn3 in 0.05% Tween 80 (FIG. 31C) was slightly reduced, withmore regions devoid of β-galactosidase activity. Negative controls forboth 7.8 mM Big CHAP (FIG. 31B) and 0.05% Tween 80 (FIG. 31D) are alsoshown.

FIG. 32A-FIG. 32F show a comparison of infiltration following Syn3administration. At lower doses of Syn3, proportionally lowerinfiltration was observed in the bladder. Decreasing concentrations ofSyn3 were used for rAd infection of bladders when using either Big CHAP(FIG. 32A, B) or Tween 80 (FIG. 32D, E). Also shown are bladders treatedwith detergent only (no Syn3) with either Big CHAP (FIG. 32C) or Tween80 (FIG. 32F).

FIG. 33A-FIG. 33D shows that administration show that administration ofSyn3 results in induction of cellular infiltrates. When the level ofinfiltration resulting from administration of virus and Syn3 (FIG. 33A)was compared to the level obtained from Syn3 alone (FIG. 33B), it wasfound that Syn3 administration results in a significant induction ofinfiltrates. Also shown are bladders from animals treated with virusonly (FIG. 33C) or a no virus/no Syn3 control (FIG. 33D).

FIG. 34 shows a pathway for synthesis of Syn3. After Reaction 3 wasconducted in DMF for 24 hours, the product was evaporated to dryness,and purified on SiO₂ with DCM/MeOH/H₂O (60:35:5).

FIG. 35 shows a protocol that was used to synthesize A-tma and A-HCl,which are analogs of Syn3 that exhibit increased solubility in aqueoussolution.

DETAILED DESCRIPTION

The present invention provides delivery enhancing compounds andformulations that enhance transport of agents into cells, such as cellspresent in epithelial tissues. The compounds and formulations canincrease the amount of an agent, such as an agent that can modulate acellular process associated with, for example, proliferation or adisease state, that enters a cell and/or increase the proportion ofcells in a tissue or organ that take up the agent. Methods of deliveringagents to cells using the delivery enhancing compounds of the inventionare also provided.

The delivery enhancing compounds and methods of the invention are usefulfor many applications that require delivery of a molecule to a cell. Forexample, diagnosis and/or treatment of many disease states oftenrequires entry of an agent into a cell that is involved in the diseaseprocess. Another example is the use of recombinant DNA technology toproduce proteins of interest, either in cell culture or in a recombinantorganism. Many additional examples of situations in which it isdesirable to introduce a compound into a cell are known to those ofskill in the art. The compounds and methods of the invention can improvethe effectiveness of each of these applications due to the increaseddelivery of an agent of interest to a target cell or tissue.

A. Delivery Enhancing Compounds

The invention provides delivery enhancing compounds that, whenformulated with an agent of interest, enhance delivery of the agent to acell. In some embodiments, the cells are present in a tissue or organ.“A delivery-enhancing compound” refers to any compound that enhancesdelivery of an agent to a cell, tissue or organ. Although anunderstanding of the mechanism by which enhanced delivery occurs is notessential to practicing the invention, it is noted that enhanceddelivery can occur by any of various mechanisms. One such mechanism mayinvolve the disruption of the protective glycosaminoglycan (GAG) layeron the epithelial surface of the tissue or organ by the deliveryenhancing compound.

Administering an agent to cells in a formulation that includes adelivery enhancing compound results in an increase in the amount ofagent that is delivered to the cells, relative to the amount of agentdelivered to the cells when administered in the absence of the deliveryenhancing compound. “Enhanced delivery” as used herein refers to eitheror both of an increase in the number of copies of an agent that entereach cell or a increase in the proportion of cells in, for example, atissue or organ, that take up the agent. In preferred embodiments, thedelivery enhancing compound results in at least about a 20% increase,more preferably at least about a 50% increase, and most preferably atleast about a 100% increase in delivery of an agent to a cell orpopulation of cells compared to the amount of the agent delivered whenadministered to cells in the absence of the delivery enhancing compound.

One can measure whether a particular compound or formulation iseffective in enhancing delivery of an agent, such as a therapeutic ordiagnostic agent, to cells by various means known to those of skill inthe art. For example, a detection reagent can be included in a deliveryenhancing formulation which is administered to the target cells. Theamount of detection reagent present in cells that are treated with thedelivery enhancing formulation is compared to that detected in cellstreated with a formulation that does not include a delivery enhancingcompound. As an example, where the agent of interest is a gene or avector that includes a gene, one can include in the formulation areporter gene for which expression is readily detectable. Where themodulating agent is a polypeptide, one can test the delivery enhancingcompounds by, for example, attaching a label to the polypeptide which ispresent in the delivery enhancing formulation and detecting the presenceand amount of label that is found in target cells after administrationof the formulation. Similarly, where molecules other than polypeptidesand polynucleotides are to be used as the modulating agent, one canlabel the molecules and detect the amount of label that enters thetarget cell population.

Examples of delivery-enhancing compounds include, but are not limitedto, detergents, alcohols, glycols, surfactants, bile salts, heparinantagonists, cyclooxygenase inhibitors, hypertonic salt solutions, andacetates. Alcohols include, for example, the aliphatic alcohols such asethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycolsinclude, for example, glycerine, propyleneglycol, polyethyleneglycol andother low molecular weight glycols such as glycerol and thioglycerol.Acetates such as acetic acid, gluconic acid, and sodium acetate arefurther examples of delivery enhancing compounds. Hypertonic saltsolutions such as 1M NaCl are also examples of delivery enhancingcompounds. Examples of surfactants include sodium dodecyl sulfate (SDS)and lysolecithin, polysorbate 80, nonylphenoxy-polyoxyethylene,lysophosphatidylcholine, polyethyleneglycol 400, polysorbate 80,polyoxyethylene ethers, polyglycol ether surfactants and DMSO. Bilesalts such as taurocholate, sodium tauro-deoxycholate, deoxycholate,chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid andother astringents like silver nitrate can also be used, as canheparin-antagonists like quaternary amines such as protamine sulfate.Cyclooxygenase inhibitors such as, for example, sodium salicylate,salicylic acid, and non-steroidal antiinflammatory drugs (NSAIDS) suchas indomethacin, naproxen, and diclofenac are also suitable.

Detergents that can function as delivery enhancing compounds include,for example, anionic, cationic, zwitterionic, and nonionic detergents.Exemplary detergents include, but are not limited to, taurocholate,deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride,ZWITTERGENT®3-14 detergent, CHAPS(3-[(3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate, hydrate,Aldrich), Big CHAP, Deoxy Big CHAP, TRITON®-X-100 detergent, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC®-F68 detergent, TWEEN® 20 detergent,and TWEEN® 80 detergent (CALBIOCHEM® Biochemicals).

One example of a preferred delivery enhancing compound for formulationsin which the agent is, for example, a nucleic acid is Big CHAP, which isa cholate derivative (see, e.g., Helenius et al. (I 979) “Properties ofDetergents” In: Methods in Enzymology, Vol.66, 734-749. In order tofacilitate the improved gene transfer for nucleic acid formulationscomprising commercial Big-CHAP preparations, the concentration of BigCHAP will vary based on its commercial source. When the Big CHAP issourced from CALBIOCHEM®, it is preferred that the concentration be in arange of 2 to 10 millimolar. More preferred is 4 to 8 millimolar. Mostpreferred is approximately 7 millimolar. When the Big CHAP is sourcedfrom Sigma, it is preferred that the concentration of Big CHAP be in arange of 15 to 35 millimolar. More preferred is 20 to 30 millimolar.Most preferred is approximately 25 millimolar.

In additional embodiments, the invention provides delivery enhancingcompounds that have a Formula I:

In Formula I, m and n can be either the same or different, and each isan integer from 2 to 8. In preferred embodiments, m and n are eachindependently 2 or 3.

R in Formula I is preferably a cationic group or a structure having theformula

Suitable cationic groups can include any moiety that will provide apositive charge to the compound. Examples of suitable cationic groupsinclude, but are not limited to, trimethylammonium and ammonium cations.

X₁ in Formula I is generally selected from the group consisting of

and X₂ and X₃ are each independently selected from the group consistingof a saccharide group,

Saccharide groups that can be used in the delivery enhancing compoundsof the invention can be monosaccharides or can include more than onemonosaccharide linked in either homo-oligosaccharides orhetero-oligosaccharides. Preferred monosaccharides include pentoseand/or hexose residues. For example, the saccharide groups can beselected from the group consisting of pentose monosaccharide groups,hexose monosaccharide groups, pentose-pentose disaccharide groups,hexose-hexose disaccharide groups, pentose-hexose disaccharide groups,and hexose-pentose disaccharide groups. One example of a preferredsaccharide group for X₃ is lactose.

In some embodiments, the delivery enhancing compounds of Formula I haveX₂ and/or X₃ as saccharide groups that are composed of three or moremonosaccharides. Preferably, the saccharide group has between one andeight monosaccharides, more preferably between one and fourmonosaccharides, and most preferably about two to three monosaccharides.The use of a trisaccharide, for example, can provide a compound havingincreased solubility.

Examples of suitable delivery enhancing compounds of the inventioninclude, but are not limited to, compounds of Formula I in which X₁ andX₂ are both

and X₃ is a saccharide.

Other embodiments have, for example, both X₁ and X₂ as

and X₃ is a saccharide group. Preferred compounds also include those inwhich n is 2 or 3, X₁ and X₂ are both

and X₃ is a hexose monosaccharide group; those in which n is 2 or 3, X₁and X₃ are both

and X₂ is a hexose monosaccharide group; and compounds in which n is 2or 3, X₁ and X₂ are both

and X₃ is a hexose-hexose disaccharide group. Also suitable arecompounds in which n is 2 or 3, X₁ and X₃ are both

and X₂ is a hexose-hexose disaccharide group, or compounds in which n is2 or 3, X₁ and X₂ are both

and X₃ is a hexose-pentose disaccharide group. Compounds of Formula Ithat have trisaccharide groups, in particular at the X₃ position, arealso preferred.

One example of a preferred delivery enhancing compound of the inventionis Syn3, which has Formula III as shown in FIG. 21. Syn3 is a syntheticanalog of an impurity that was found in commercial preparations of BigCHAP (see, Examples). Impurities 2 and 3 of Big CHAP are also suitablefor use as delivery enhancing compounds, particularly when formulated ina solubilizing buffer that contains, for example, a detergent such asBig CHAP.

For some applications, it is desirable to use delivery enhancingcompounds that exhibit increased water solubility and/or deliveryenhancing activity compared to other compounds. Such compounds areprovided by the invention. For example, the invention provides compoundsthat have the Formula I in which R is a cationic group. Suitablecationic groups include, for example, tetramethyl and ammonium moieties,and salts thereof. Examples of such compounds include A-tma (Formula IV)and A-HCl (Formula V) as shown in FIG. 21. Other compounds with improvedsolubility and/or delivery enhancing activity include those in which thesaccharide group or groups in compounds of Formula I are trisaccharidesor longer.

In some embodiments, the delivery enhancing agents of the presentinvention have the Formula II:

wherein X₁ and X₂ are selected for the group consisting of

and X₃ is a saccharide group. Suitable saccharide groups include thosediscussed above for compounds of Formula I. In one example of a suitablecompound, both X₁ and X₂ are

and X₃ is a glucose group, Additional examples of suitable compoundsinclude, but are not limited to, those in which both X₁ and X₂ areselected from the group consisting of

and X₃ is a lactose group.

The invention also provides formulations that contain an agent to bedelivered to a cell and a delivery enhancing compound. The concentrationof the delivery enhancing compound in a formulation will depend on anumber of factors such as the particular delivery enhancing compoundbeing used, the buffer, pH, target tissue or organ and mode ofadministration. The concentration of the delivery enhancing compoundwill often be in the range of 1% to 50% (v/v), preferably 10% to 40%(v/v) and most preferably 15% to 30% (v/v). The delivery enhancingcompounds of the invention are preferably used in the range of about0.002 to 2 mg/ml, more preferably about 0.02 to 2 mg/ml, most preferablyabout 0.1 to 1 mg/ml in the formulations of the invention.

The delivery enhancing compounds of the invention are typicallyformulated in a solvent in which the compounds are soluble, althoughformulations in which the compounds are only partially solubilized arealso suitable. Phosphate buffered saline (PBS) is one example of asuitable solubilizing agent for these compounds, and others are known tothose of skill in the art. One will recognize that certain additionalexcipients and additives may be desirable to achieve solubilitycharacteristics of these agents for various pharmaceutical formulations.For example, well known solubilizing agents such as detergents, fattyacid esters, surfactants can be added in appropriate concentrations soas to facilitate the solubilization of the compounds in the varioussolvents to be employed. Where the formulation includes a detergent, thedetergent concentration in the final formulation administered to apatient is preferably about 0.5-2X the critical micellizationconcentration (CMC). Suitable detergents include those listed above. Theidentification of suitable detergents and appropriate concentrations fortheir use can be determined as described herein.

One example of a preferred solubilizing agent for compounds such as Syn3and related compounds is Tween 80 at a concentration of approximately0.05% to about 0.3%, more preferably at a concentration of about 0.10%to about 0. 15%. Big CHAP is also a preferred solubilizing agent forSyn3 and related compounds.

The compounds of the invention may be used alone, in combination witheach other, or in combination with another delivery-enhancing agent.

B. Modulatory Agents

The delivery-enhancing compounds of the invention are useful forenhancing the delivery of agents, including proteins, nucleic acids,antisense RNA, small molecules, and the like, to cells. For example, thedelivery enhancing compounds are useful for delivering agents to cellsthat are part of any tissue or organ, including those that have anepithelial membrane.

Among the agents that are suitable for delivery using the deliveryenhancing compounds are “modulatory agents,” which, as used herein,refers to agents that can modulate biological processes. Such processesinclude, for example, cell growth, differentiation, proliferation(including neoplastic disorders such as cancer), regulation, metabolicor biosynthetic pathways, gene expression, and the like. Modulatoryagents can also influence, for example, immune responses (includingautoimmune disorders), infection by bacterial and fungal pathogens, andany other biological process that is regulatable by introduction of amodulatory agent.

Therapeutic agents are an example of modulatory agents that one candeliver using the delivery-enhancing agents. Such agents are useful formodulating cellular processes that are associated with disease. The term“therapeutic agent” as used herein includes but is not limited totherapeutic proteins, therapeutic genes, vectors (plasmid or viralvectors) containing a therapeutic gene, antisense nucleic acids, orother therapeutic nucleic acid sequences (e.g., triplex nucleic acids).For purposes of the present invention, the term “therapeutic gene,”refers to a nucleic acid sequence introduced into a cell to achieve atherapeutic effect. Examples of such therapeutic genes include, but arenot limited to, tumor suppressor genes, suicide genes, antisense nucleicacid molecules, triplex forming nucleic acid molecules, genes encodingcytokines (such as but not limited to the interferons α, β, δ, and γ),genes encoding interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-7 andIL-10), and colony stimulating factors such as GM-CSF. In someinstances, the therapeutic gene may present in a naturally occurring orrecombinantly modified virus.

A suicide gene is a nucleic acid sequence, the expression of whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell. A well known example of a suicide gene isthe thymidine kinase (TK) gene (see, e.g., Woo et al., U.S. Pat. No.5,631,236, issued May 20, 1997; Freeman et al., U.S. Pat. No. 5,601,818,issued Feb. 11, 1997) in which the cells expressing the TK gene productare susceptible to selective killing by the administration ofgancyclovir.

Antisense nucleic acid molecules are complementary oligonucleotidestrands of nucleic acids designed to bind to a specific sequence ofnucleotides to inhibit production of proteins, including disease-causingproteins. Antisense molecules which bind to specific oncogenes arefrequently used to inhibit the transcription of these cancer causingagents. These agents can be used alone or in combination with othertherapeutic genes.

Triplex forming nucleic acids are molecules designed to inhibittranscription of genes, including, for example, disease causing genes.Generally, this is achieved by the triplex forming nucleic acid bindingto the transcriptional control sequence of the target gene andpreventing the transcription of the target gene. Triplex formingoligonucleotides recognize and bind to the major groove ofdouble-stranded DNA by virtue of Hoogsteen hydrogen bonding. Examples ofthe use of triplex technology include targeting of the androgen receptoror the insulin-like growth factor genes with triplex technology inprostate cancer cells. Boulikas, T., Anticancer Res. 17(3A): 1471-1505(1997). Triplex nucleic acids have been demonstrated to be mutagenic insome instances and such molecules may be used to induce responses ofendogenous DNA repair mechanisms leading to an induction of tumorsuppressor genes in a therapeutic manner and may contribute to genomicinstability inducing apoptosis in the target cell. A variety of triplexnucleic compounds are currently under investigation and are welldocumented in the scientific literature.

“Tumor suppressor gene” refers to a gene which encodes a polypeptidethat suppresses the formation of tumors. Tumor suppressor genes arenaturally occurring genes in mammalian cells the deletion orinactivation of which is believed to be a necessary prerequisite fortumor development. Tumor suppressor gene therapy generally attempts toreintroduce the tumor suppressor gene to cells in which the gene isabsent or inactive. Examples of tumor suppressor genes useful in thepractice of the present invention include p53, p110Rb, members of theINK4 family of tumor suppressor genes including p16 and p21 andtherapeutically effective fragments thereof such as p56Rb, p94Rb, etc.In the preferred practice of the invention, the tumor suppressor gene isselected from the Rb gene and the p53 gene and nucleic acid sequencesencoding functional variants thereof, such as Rb56. In the mostpreferred practice of the invention, the tumor suppressor gene is p53.

In some embodiments, the compositions of the invention comprise a“therapeutically effective” amount of a therapeutic agent in a buffercomprising a delivery-enhancing compound. “Therapeutically effective” asused herein refers to the prevention of, reduction of, or curing ofsymptoms associated with a disease state.

The delivery-enhancing agents and formulations that contain these agentscan also be used to facilitate delivery of genes of interest to cells,in particular cells of organs and tissues. These genes can encode, forexample, proteins that are of interest for commercial purposes. As anexample, one can use the agents and formulations to deliver to mammarytissue of a mammal a gene that encodes a nutritionally important proteinwhich is then secreted in the milk produced by the mammal. Other uses ofsuch agents and formulations will be evident to those of skill in theart.

The delivery enhancing agents and formulations that include such agentsare also useful for delivering diagnostic agents to cells, organs andtissues. Examples of diagnostic agents include marker genes that encodeproteins that are readily detectable when expressed in a cell(including, but not limited to, β-galactosidase, green fluorescentprotein, luciferase, and the like) and labeled nucleic acid probes(e.g., radiolabeled probes).

C. Vectors for Gene Delivery

In the situation where an agent to be delivered to a cell is a gene, onecan incorporate the gene into a vector. Examples of vectors used forsuch purposes include expression plasmids capable of directing theexpression of the gene of interest in the target cell. In otherinstances, the vector is a viral vector system wherein the gene ofinterest is incorporated into a viral genome capable of transfecting thetarget cell. Where the gene of interest is designed for expression in atarget cell, the gene can be operably linked to expression and controlsequences that can direct expression of the gene in the desired targethost cells. Thus, one can achieve expression of the gene underappropriate conditions in the target cell.

Viral vector systems useful in the practice of the instant inventioninclude, for example, naturally occurring or recombinant viral vectorsystems. Depending upon the particular application, suitable viralvectors include replication competent, replication deficient, andconditionally replicating viral vectors. For example, viral vectors canbe derived from the genome of human or bovine adenoviruses, vacciniavirus, herpes virus, adeno-associated virus, minute virus of mice (MVM),HIV, sindbis virus, and retroviruses (including but not limited to Roussarcoma virus), and MoMLV. Typically, genes of interest are insertedinto such vectors to allow packaging of the gene construct, typicallywith accompanying viral DNA, infection of a sensitive host cell, andexpression of the gene of interest. A preferred recombinant viral vectoris the adenoviral vector delivery system which has a deletion of theprotein IX gene (see, International Patent Application WO 95/11984,which is herein incorporated by reference in its entirety for allpurposes).

“Recombinant” as used herein refers to nucleic acids and the proteinsencoded by them wherein the nucleic acids are constructed by methods ofrecombinant DNA technology, also termed “genetic engineering.”

Therapeutically effective amounts of the pharmaceutical compositioncomprising a modulatory gene, such as a p53 gene or a retinoblastomatumor suppressor gene, in a recombinant viral vector delivery systemformulated in a buffer comprising a delivery-enhancing agent, will beadministered in accord with the teaching of this invention. For example,therapeutically effective amounts of a therapeutic gene in therecombinant adenoviral vector delivery system formulated in a buffercontaining a delivery-enhancing agent are in the range of about 1×10⁸,particles/ml to 1×10¹² particles/ml, more typically about 1×10⁸particles/ml to 5×10¹¹ particles/ml, most typically 1×10⁹ particles/mlto 1×10¹¹ particles/ml (PN/ml).

D. Gene Delivery Systems

As used herein, “gene delivery system” refers to any means for thedelivery of an agent to a target cell. The agent can be associated witha gene delivery system which is then delivered to the cell using aformulation that contains a delivery enhancing compound.

In some embodiments of the invention, gene constructs or other agentsare conjugated to a cell receptor ligand for facilitated uptake (e.g.,invagination of coated pits and internalization of the endosome) throughan appropriate linking moiety, such as a DNA linking moiety (Wu et al.,J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180). For example, geneconstructs can be linked through a polylysine moiety toasialo-oromucocid, which is a ligand for the asialoglycoprotein receptorof hepatocytes.

Similarly, viral envelopes used for packaging gene constructs can bemodified by the addition of receptor ligands or antibodies specific fora receptor to permit receptor-mediated endocytosis into specific cells(see, e.g., WO 93/20221, WO 93/14188, WO 94/06923). In some embodimentsof the invention, the DNA constructs of the invention are linked toviral proteins, such as adenovirus particles, to facilitate endocytosis(Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88: 8850-8854 (1991)). Inother embodiments, molecular conjugates of the instant invention caninclude microtubule inhibitors (WO/9406922); synthetic peptidesmimicking influenza virus hemagglutinin (Plank et al., J. Biol. Chem.269:12918-12924 (1994)); and nuclear localization signals such as SV40 Tantigen (WO93/19768).

In some embodiments of the invention, the modulating agent is anantisense nucleic acid. The antisense nucleic acid can be provided as anantisense oligonucleotide (see, e.g., Murayama et al., Antisense NucleicAcid Drug Dev. 7:109-114 (1997)). Genes encoding an antisense nucleicacid can also be provided; such genes can be formulated with a deliveryenhancing compound and introduced into cells by methods known to thoseof skill in the art. For example, one can introduce a gene that encodesan antisense nucleic acid in a viral vector, such as, for example, inhepatitis B virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173(1997)); in adeno-associated virus (see, e.g., Xiao et al., Brain Res.756:76-83 (1997));

or in other systems including, but not limited, to an HVJ (Sendaivirus)-liposome gene delivery system (see, e.g., Kaneda et al., Ann.N.Y. Acad. Sci. 811:299-308 (1997)); a “peptide vector” (see, e.g.,Vidal et al., CR Acad. Sci III 32:279-287 (1997)); as a gene in anepisomal or plasmid vector (see, e.g., Cooper et al., Proc. Natl. Acad.Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584(1997)); as a gene in a peptide-DNA aggregate (see, e.g., Niidome etal., J. Biol. Chem. 272:15307-15312 (1997)); as “naked DNA” (see, e.g.,U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466); in lipidic vectorsystems (see, e.g., Lee et al., Crit Rev Ther Drug Carrier Syst.14:173-206 (1997)); polymer coated liposomes (Marin et al., U.S. Pat.No. 5,213,804, issued May 25, 1993; Woodle et al., U.S. Pat. No.5,013,556, issued May 7, 1991); cationic liposomes (Epand et al., U.S.Pat. No. 5,283,185, issued Feb. 1, 1994; Jessee, J. A., U.S. Pat. No.5,578,475, issued Nov. 26, 1996; Rose et al, U.S. Pat. No. 5,279,833,issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No. 5,334,761, issuedAug. 2, 1994); gas filled microspheres (Unger et al., U.S. Pat. No.5,542,935, issued Aug. 6, 1996), ligand-targeted encapsulatedmacromolecules (Low et al. U.S. Pat. No. 5,108,921, issued Apr. 28,1992; Curiel et al., U.S. Pat. No. 5,521,291, issued May 28, 1996;Groman et al., U.S. Pat. No. 5,554,386, issued Sep. 10, 1996; Wu et al.,U.S. Pat. No. 5,166,320, issued Nov. 24, 1992).

E. Pharmaceutical Formulations

When used for pharmaceutical purposes, the formulations of the inventioninclude a buffer that contains the delivery-enhancing compound. Thebuffer can be any pharmaceutically acceptable buffer, such as phosphatebuffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycinebuffer, sterile water, and other buffers known to the ordinarily skilledartisan such as those described by Good et al. (1966) Biochemistry5:467. The pH of the buffer in the pharmaceutical composition comprisinga modulatory gene contained in an adenoviral vector delivery system, forexample, is typically in the range of 6.4 to 8.4, preferably 7 to 7.5,and most preferably 7.2 to 7.4.

The compositions of this invention can additionally include astabilizer, enhancer or other pharmaceutically acceptable carriers orvehicles. A pharmaceutically acceptable carrier can contain aphysiologically acceptable compound that acts, for example, to stabilizethe recombinant adenoviral vector delivery system comprising the tumorsuppressor gene. A physiologically acceptable compound can include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Otherphysiologically acceptable compounds include wetting agents, emulsifyingagents, dispersing agents or preservatives, which are particularlyuseful for preventing the growth or action of microorganisms. Variouspreservatives are well known and include, for example, phenol andascorbic acid. One skilled in the art would know that the choice ofpharmaceutically acceptable carrier depends on the route ofadministration and the particular physio-chemical characteristics of therecombinant adenoviral vector delivery system and the particular tumorsuppressor gene contained therein. Examples of carriers, stabilizers oradjuvants can be found in Martin, Remington's Pharm. Sci., 15th Ed.(Mack Publ. Co., Easton, Pa. 1975), which is incorporated herein byreference.

F. Administration of Formulations

In some embodiments, the delivery-enhancing compound is included in thebuffer in which the modulating agent is formulated. Thedelivery-enhancing compound can be administered prior to the modulatingagent or concomitant with the modulating agent. In some embodiments, thedelivery-enhancing compound is provided with the modulating agent bymixing a modulating agent preparation with a delivery-enhancing compoundformulation just prior to administration to the patient. In otherembodiments, the delivery-enhancing compound and modulating agent areprovided in a single vial to the caregiver for administration.

In the case of a pharmaceutical composition comprising a tumorsuppressor gene contained in a recombinant adenoviral vector deliverysystem formulated in a buffer which further comprises adelivery-enhancing agent, the pharmaceutical composition can beadministered over time in the range of about 5 minutes to 3 hours,preferably about 10 minutes to 120 minutes, and most preferably about 15minutes to 90 minutes. In another embodiment the delivery-enhancingagent may be administered prior to administration of the recombinantadenoviral vector delivery system containing the tumor suppressor gene.The prior administration of the delivery-enhancing agent may be in therange of about 30 seconds to 1 hour, preferably about 1 minute to 10minutes, and most preferably about 1 minute to 5 minutes prior toadministration of the adenoviral vector delivery system containing thetumor suppressor gene.

The modulating agent formulated in a buffer comprising adelivery-enhancing agent can be delivered to any tissue or organ,including neoplastic tissues such as cancer tissue, using any deliverymethod known to the ordinarily skilled artisan for example, intratumoralor intravesical administration. Tissues and organs include any tissue ororgan having an epithelial membrane such as the gastrointestinal tract,the bladder, respiratory tract, and the lung. Examples include but arenot limited to carcinoma of the bladder and upper respiratory tract,vulva, cervix, vagina or bronchi; local metastatic tumors of theperitoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma;carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose,larynx, oesophagus, stomach, colon and rectum, gallbladder, or skin; ormelanoma.

In some embodiments of the invention, the therapeutic agent isformulated in mucosal, topical, and/or buccal formulations, particularlymucoadhesive gel and topical gel formulations. Exemplary permeationenhancing compositions, polymer matrices, and mucoadhesive gelpreparations for transdermal delivery are disclosed in U.S. Pat. No.5,346,701. Such formulations are especially useful for the treatment ofcancers of the mouth, head and neck cancers (e.g., cancers of thetracheobronchial epithelium) skin cancers (e.g., melanoma, basal andsquamous cell carcinomas), cancers of the intestinal mucosa, vaginalmucosa, and cervical cancer.

In some embodiments of the invention, a therapeutic agent is formulatedin ophthalmic formulations for administration to the eye. Suchformulations are useful in the delivery of the retinoblastoma (RB) geneto the eye, optionally in conjunction with the delivery of p53.

G. Methods of Treatment

The formulations of the invention are typically administered to enhancetransfer of an agent to a cell. The cell can be provided as part of atissue, such as an epithelial membrane, or as an isolated cell, such asin tissue culture. The cell can be provided in vivo, ex vivo, or invitro.

The formulations containing delivery enhancing compounds and modulatingagents can be introduced into the tissue of interest in vivo or ex vivoby a variety of methods. In some embodiments of the invention, themodulating agent is introduced to cells by such methods asmicroinjection, calcium phosphate precipitation, liposome fusion, orbiolistics. In further embodiments, the therapeutic agent is taken updirectly by the tissue of interest.

In some embodiments of the invention, the compositions of the inventionare administered ex vivo to cells or tissues explanted from a patient,then returned to the patient. Examples of ex vivo administration oftherapeutic gene constructs include Arteaga et al., Cancer Research56(5):1098-1103 (1996); Nolta et al., Proc Natl. Acad. Sci. USA93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65(1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandroet al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov etal., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).

EXAMPLES

The following examples are intended to illustrate, not limit the scopeof this invention. In the following examples, “g” means grams, “ml”means milliliters, “mol” means moles, “° C.” means degrees Centigrade,“min.” means minutes, “DMF” means dimethylformamide, and “PN” specifiesparticle number. All temperatures are in degrees Centigrade unlessotherwise specified.

Example 1 Ethanol Improves Gene Transfer in the Bladder

Initial experiments have shown that several factors including virusconcentration, time of administration, and volume of dosing caninfluence gene transfer to the bladder epithelium after intravesicaladministration to rats. Because increased penetration of dyes can beachieved by intravesical administration of different solvents,modification of the adenovirus formulation was also investigated as analternative strategy to increase adenovirus transgene expression in thebladder (Monson et al., Urology 145:842-845 (1991)). The instantexperiments focused on the use of ethanol to increase adenovirustransgene expression in the bladder.

Nine female buffalo rats (Harlan Sprague Dawley) were anesthetized withisoflurane and received a single intravesical administration of a humanrecombinant adenovirus encoding the lacZ gene (rAd-βgal). The humanrecombinant adenoviral vector comprising the lacZ gene (rAd-βgal) isdescribed in Wills et al., Human Gene Therapy 5:1079-1088 (1994). Beforeinstallation bladders were flushed with PBS and emptied. rAd-βgal wasthen diluted to achieve a final concentration of 1.7×10¹¹ PN/mL in 1)VPBS (2% (w/v) sucrose and 2 mM MgCl, in PBS), 2) 30% (v/v) ethanol, or3) 50% (v/v) DMSO, and instilled in a 250 μL volume (N=3 animals/group).The administered material was retained in the bladder for 45 minutes.The bladder were then flushed with PBS, and the animals were permittedto recover from the procedure. Two days after administration, rats weresacrificed, bladders were harvested, fixed, and whole organs werestained with an Xgal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside)solution to evaluate reporter gene transfer. Xgal-stained tissues werethen paraffin embedded, sectioned, and counter stained with hematoxylinand eosin. Hydrolysis of Xgal by β-galactosidase results in a blue colorthat localized to the superficial luminal bladder epithelium.

Transgene expression, consequent to delivery by the adenoviral vector,was detected in bladders from all animals treated with rAd-βgal but notin an untreated control. Transgene expression was similar to previouslypublished results using the PBS/sucrose formulation (Bass et al., CancerGene Therapy 2:2:97-104 (1995)). In sharp contrast, β-galactosidaseexpression in the luminal epithelial surface was greatly enhanced inanimals that received rAd-βgal diluted in 30% ethanol (FIG. 1). Bladderspecimens described in FIG. 1 were embedded, sectioned, andcounterstained with hematoxylin and eosin. Histologic evaluation of thebladder tissue demonstrated increased β-galactosidase expression of thetransitional bladder epithelium when ethanol was added to the adenovirusformulation (FIG. 2). The interaction of ethanol with the protectiveglycosaminoglycan (GAG) layer on the epithelium surface provides amechanism for the observed increase in transgene expression. Disruptionof this layer may facilitate virus-cell interaction at the surface andpotentially enhance penetration into the submucosa.

Example 2 Dose-Dependent Transgene Expression in the Rat Bladder

In another experiment, 18 female Sprague-Dawley rats were anaesthetizedwith isoflurane and received a single 0.5 ml intravesical bolus ofrAd-βgal at concentrations of 2×10⁷, 2×10⁸, 2×10⁹, 2×10¹⁰, and 2×10¹¹,PN/mL in a 22.5% (v/v) ethanol formulation. After a 45 minuteincubation, the bladders were flushed with PBS, and animals werepermitted to recover from anesthesia. Two days later, animals weresacrificed, and bladders were harvested, fixed, and whole organs werestained with Xgal solution to evaluate adenovirus transgene expression.β-galactosidase expression in the luminal bladder epithelium correlatedwith the concentration of the administered recombinant adenovirus (FIG.3). No striking differences were observed among animals receiving 2×10¹⁰or 2×10¹¹ PN/mL, suggesting a saturation of transgene expression in thismodel. Analysis of the volume voided after instillation indicated only aminimal reduction in the infectious titer of the dosing material atthese high doses. Expression of β-galactosidase decreased at lowerconcentrations. No evidence of β-galactosidase expression was detectedin animals dosed at a concentration of 1×10⁷ PN/mL or in an untreatedcontrol animal.

Example 3 ACNRB Gene Transfer in the Mouse Bladder

A pilot study was conducted to specifically evaluate expression of theRB transgene using a RT-PCR assay. The recombinant adenovirus used inthis study was based on serotype 5 human adenovirus from which the viralearly region 1 encoding E1a, E1b, and pIX proteins have been deleted.This adenovirus is limited to propagation in 293 cells which produce theAd5 E1 gene products required for replication. Transfer plasmidsencoding either full length or truncated Rb were generated from pACN(Wills et al., Cancer Gene Therapy 2:191-197 (1995)) and were, in turn,used to construct the recombinant adenoviruses. Either a full-length RBcDNA (1-928 amino acids), subcloned as a 2.8 Kb Xba I—Bam HI fragmentfrom the plasmids pETRbc (Huang et al., Nature 350:160-162 (1991) or atruncated fragment (amino acids 381-928), subcloned as a 1.7 KB XbaI—Bam HI cDNA fragment, was placed downstream of the CMVpromoter/enhancer and the Ad 2 tripartite leader cDNA of the plasmidpACN. These plasmids were subsequently linearized with Eco RI andcotransfected (CaPO₄, Stratagene) with either the isolated Cla Idigested large fragment of H5ilE4 (Hemstrom et al., J. Virol.62:3258-3264 (1988)), to make Ad-RB56 (ACN56) containing a partial E4deletion, or with the large fragment from a hybrid virus of dl327(Ginsberg et al. Proc. Natl. Acad. Sci. U.S.A. 86:3823-3827 (1989)) andH5ilE4 to create Ad-Rb110 (ACNRB) which contains deletions in both theE3 and E4 regions of the vector.

Eight female ICR mice (Charles River Laboratories) were anesthetizedwith avertine and each received a single 80 μl intravesicaladministration of (ACNRB). ACNRB (4×10¹¹ PN/mL) was diluted and preparedin a PBS solution or a 30% (v/v) ethanol solution. After the virus wasretained in the bladder for 45 minutes, the animals were permitted torecover and void. Mice were sacrificed 2 days or 14 days after ACNRBadministration, and bladders, livers, and kidneys from each animal wereharvested, homogenized, and processed for analysis (N=2 animals/group).Transgene expression was determined using RT-PCR with a primer specificfor ACNRB. More specifically, primers were generated to identify ACNRBand amplify the region from the 3′ end of the CMV sequence and to the 5′end of the RB sequence. Following amplification (30 cycles) RT-PCRproducts were separated on a 10% polyacrylamide gel, stained withethidium bromide, and photographed. Increased ACNRB expression wasdetected after treatment with ACNRB in 30% (v/v) ethanol compared tovery low expression after treatment with ACNRB in VPBS. Positivecontrols for the assay included samples from ACNRB-infected 5637 humanbladder cancer cells (CONTROL). Bladder RNA samples from ACNRB-infectedanimals that were amplified with primers specific for beta-actinprovided an internal control for the quality of RNA. Untreated samplesand bladder samples without the reverse transcriptase (RT) providedcontrols for contaminating DNA. Two days after dosing, levels of ACNRBexpression in the bladder homogenates were detected from animals thatreceived ACNRB prepared in 30% ethanol (FIG. 4). No evidence ofexpression was detected in non-bladder tissue or in any samplescollected 14 days after dosing.

Example 4 Kinetics of Biodistribution and ACNRB Expression AfterIntravesical Administration to Mice

To investigate the time course of expression after intravesicaladministration, 40 female mice (Charles River Laboratories) wereanaesthetized with avertine and received a single 80 μL bolus of ACNRB(4×10¹⁰ PN/mL in 22% (v/v) ethanol). The instilled material was retainedin the bladder for approximately 45 minutes, and animals were permittedto recover from the procedure. Mice were sacrificed 1, 2, 3, 4, 5, 6, 7,and 14 days after administration (N=4/time) for analysis. Bladders,livers, and kidneys were harvested and snap frozen in liquid nitrogenfor subsequent analysis. For detection of ACNRB expression, tissuesamples were homogenized, and total RNA was extracted usingTRI-Reagent®. An aliquot of total RNA was amplified in an RT-PCR assayusing primers specific for ACNRB to distinguish transgene expressionfrom endogenous RB expression. For detection of ACNRB DNA, a DNAextraction kit (Stratagene) was used on tissue homogenates. PCR wasperformed with the primers specific for ACNRB, as described above forthe RT-PCR analysis.

ACNRB transgene expression in the bladder homogenates was detected onlyin samples collected on days 1-6, with expression relative to endogenousp53 decreasing with time (FIG. 5, upper panel). No expression wasdetected from samples collected 7 and 14 days after administration.Interestingly, some ACNRB expression was detected in the kidneys on days1, 2 and 3, but no expression was observed in the liver (FIG. 5, lowerpanels).

ACNRB DNA was detected in bladder tissue of all animals that receivedACNRB, including those harvested 14 days after administration (FIG. 6,(left panel)). DNA was also recovered from the kidney homogenates,consistent with the ACNRB expression detected in this tissue (FIG. 6,right panel). No evidence for ACNRB DNA was detected in liver samplesharvested during the study (data not shown). Samples from an untreatedanimal (U) and purified ACNRB DNA (PC) were used as negative and 25positive controls, respectively.

Because systemic administration of recombinant adenovirus resultsprimarily in transgene expression in the liver (Li et al., Human GeneTherapy 4:403-409 (1993)), the absence of ACNRB DNA and expression inliver samples (FIG. 5 and FIG. 6) suggests negligible systemic exposureof ACNRB after intravesical administration. Retrograde flow via theureters may have contributed to the detection of ACNRB in the kidney.

The data presented above demonstrate transgene expression in the rodentbladder following intravesical administration of ACNRB. These studiesfurther indicate that adenovirus-mediated gene transfer to the bladderepithelium can be enhanced by the presence of a delivery-enhancingagent, such as ethanol, in the formulation. One mechanism for theincreased gene transfer may be the disruption of the protectiveglycosaminoglycan layer on the epithelial surface of the bladder. Asingle intravesical administration of ACNRB in a 20-30% (v/v) ethanolformulation results in transgene expression in the bladder that persistsfor. approximately one week. Retrograde ureteral flow provides a likelyexplanation for the transient expression of ACNRB detected in thekidney. The absence of ACNRB expression and ACNRB DNA in the liverindicates limited systemic exposure after intravesical administration.

Example 5 Use of Detergent Formulations

To minimize side effects without losing gene transfer efficiency, otherexcipients were tested. Detergents are known to interact with cellmembranes and form large pores without further damaging the cells. Theefficiency of recombinant adenovirus formulated in less toxic detergentswas studied in rats and mice gene transfer models.

rAd-βgal was formulated in different detergents at their criticalmicellization concentration to evaluate efficiency of gene transfer tothe bladder epithelium. Female rats (about 200 g b/w, Harlan SpragueDawley) were anesthetized with isoflurane and received a singleintravesical administration of rAd-βgal (1×10¹¹ PN/ml) in differentdetergent formulations (see Table I). Before instillation, bladders wereflushed with PBS and then emptied. rAd-βgal was then instilled in avolume of 0.5 ml. The instilled solution was retained in the bladder for45 minutes. The bladders were then flushed with PBS, and the animalswere permitted to recover from the procedure. 48 hours afteradministration, the rats were sacrificed, the bladders harvested, andfixed in formalin. After fixation, the bladders were openedlongitudinally so that the urothelium was exposed to the chromogen(Xgal), that is converted to a blue color, if reporter gene(β-galactosidase) expression is present. The luminal epithelial surfaceof the whole bladder was photographed an blue staining scored: +(minimal staining), ++ (moderate staining), +++ intense stainingcovering the whole bladder epithelial surface. The results are shown inTable I. Some of the anionic detergents (taurodeoxycholate),zwitterionic detergents (CHAPS, ZWITTERGENT®, and non-ionic detergents(Big CHAP (CALBIOCHEM®), TRITON® X-100) enhanced gene transferdramatically. Cationic detergents and some of the nonionic detergents(PLURONIC®D F68, TWEEN®), did not have similar effects. Improvements ofgene transfer were often accompanied by cystitis. Zwiterionic detergentsfacilitated bladder stone formation.

Possible manifestations of cystitis as observed with ethanol wereevaluated in mice using a 7 mM Big CHAP (CALBIOCHEM®) (2X CMC) or 0.05mM TRITON®-X-100 detergent (CMC) formulation. The formulations wereadministered intravesically in a volume of 80 uL, and animals wereobserved over a 7-day interval. After sacrifice, bladders wereparaffin-embedded, sectioned, and stained with hematoxylin and eosin forpathologic evaluation. Only a slight macrophage infiltration into thebladder tissue was observed in mice treated with Big CHAP (CALBIOCHEM®).Macrophages infiltrated more prominently (slight to mild) induced byTRITON®-X-100 detergent. In sharp contrast, significant cystitis wasdetected in animals treated with 22% ethanol.

Example 6 Gene Transfer of ACNRB

In addition to the experiments with the reporter gene, a different setof studies was conducted to specifically evaluate gene transfer ofACNRB. Female ICR mice were anesthetized with avertine and each mousereceived a single 80 μL intravesical administration of ACNRB. ACNRB(4×10¹⁰ PN/mL) was formulated in VPBS, 22% (v/v) ethanol, or 3 mM BigCHAP (CALBIOCHEM®). After the virus was retained in the bladder for 45minutes, the animals were permitted to recover. Mice were sacrificed 48hours after ACNRB administration, and bladders snap frozen in liquidnitrogen. Transgene expression was determined using RT-PCR. Tissues wererinsed in RNAse free water, homogenized, digested in Tri-Reagent(Molecular Research Center), and total cellular RNA extracted. ACNRB wasprobed using a 5′ primer located in the CMV region of ACNRB vector, anda 3′ primer resided in the 5′ end of Rb genome. RT-PCR was performed inthe Perkin Elmer 9600 GeneAmp PCR System. Cycling conditions were 10 minat 65° C., 8 min at 50° C., 5 min at 95° C. 32 cycles of PCR wereperformed, each cycle consisting of 30 sec at 94° C., 30 sec at 58° C.,and 30 sec at 72° C. The 32nd cycle included a 10 min elongation step at72° C. to ensure full extension of incomplete DNA fragments. ACNRB-RNAbands were stained with ethidium bromide. The results, enhancedexpression using an ethanol or Big CHAP (CALBIOCHEM®) formulation, areshown in FIG. 9.

Example 7 Big CHAP (CALBIOCHEM®) Enhances Transgene Expression withMinimal Cystitis

Because Big CHAP (CALBIOCHEM®) enhanced gene transfer with minimalcystitis, this formulation was selected for further evaluation,including concentration and dose-dependence in studies similar to thosedescribed above. Briefly, in anaesthetized female rats rAd-βgal (1×10¹¹PN/ml) was administered into the bladder via an intravesical catheter.rAd-βgal was formulated in different concentrations of Big CHAP(CALBIOCHEM®). A volume of 0.5 ml was injected and remained instilled inthe bladder for 45 minutes. The animals were sacrificed 48 hours later,the bladder fixed in 4% formalin/glutaraldehyde, opened longitudinally,and the β-galactosidase enzyme activity measured using Xgal substrate.The intensity of blue staining correlates with the βgal-transgeneexpression. FIG. 7 shows the epithelial surface of Xgal stainedbladders. The results indicate a concentration-dependent increase ofgene transfer to the epithelium. The 3.5-7 mM concentrations of Big CHAP(CALBIOCHEM®) significantly improved gene transfer. The formulationalone (FIG. 7, lower panel) did not induce a blue color from the Xgalsubstrate. A higher concentration (17.5) mM did not notably improve genetransfer or expression, but induced cystitis in some of the animalstested.

TABLE I Dose Gene Expression in Excipient Charge of Detergent (mM)Bladder Epithelium Gross Pathology Stability Taurocholate anionic 6 +none ND Deoxycholate anionic 5 + Cystitis ND Taurodeoxycholate anionic 6+++ Cystitis + Cetylpyridinium cationic 0.9 + none − BenzalkoniumChloride cationic 0.5% <+ none − Zwittergent ® 3-14 zwitterionic 4 +++stone formation ND Chaps zwitterionic 7 +++ stone formation + Big CHAP(CALBIOCHEM ®) non ionic 3.5 +++ none + Deoxy Big CHAP non ionic 1.5 +++Cystitis ND (CALBIOCHEM ®) Triton X-100 non ionic 0.05 +++ none + C12E8non ionic 4 ++ none ND Octyl-β-D-Glucopyranoside non ionic 10 ++ none NDPluronic F68 non ionic 0.04 + none + Tween 20 non ionic 2 + none + Tween80 non ionic 0.02 + none ND Tween 80 non ionic 2 + none +

Effects of higher recombinant adenovirus concentrations were alsotested. Briefly, in anaesthetized female rats different concentrationsof rAd-βgal, formulated in 7 mM Big CHAP (CALBIOCHEM®) were administeredinto the bladder via an intravesical catheter. The animals weresacrificed 48 hours later, the bladder fixed in 4%formalin/glutaraldehyde, opened longitudinally, and Xgal stained. FIG. 8shows a concentration dependent increase of gene transfer to theepithelium. A concentration of 1.3×10¹¹ PN/ml induced maximal genetransfer. A higher concentration (6.5×10¹¹ PN/ml) did not notablyimprove the blue staining. In lower concentrations of rAd-βgal, 1.3×10¹⁰PN/ml, or 1.3×10⁹ PN/ml, transgene expression reduced dose dependently.When 3.5 mM and 7 mM formulations were compared, β-galactosidaseexpression was similar, although the enhanced effect appeared morereproducible in animals treated with the 7 mM Big CHAP (CALBIOCHEM®)formulation.

Example 8 Transgene Expression in Tumors with Big CHAP (CALBIOCHEM®D)Formulation

Because initial investigations focused on animals with intact bladderepithelium, evaluated adenovirus mediated gene transfer in an animalmodel of transitional cell carcinoma was also studied. Tumors wereinduced in male Fisher rats by addition of 0.05% BBN in the drinkingwater for six months. rAd-βgal (1×10¹¹ PN/ml), formulated in 4 mM BigCHAP (CALBIOCHEM®) or VPBS was instilled into the bladder for 45 minutesby direct injection. β-gal expression was evaluated 48 hr aftertreatment. Consistent with earlier experiments using non-tumor bearinganimals, gene transfer to tumor tissue was improved with the Big CHAP(CALBIOCHEM®) formulation compared to the VPBS formulation (FIG. 10).

Gene transfer of rAd carrying the p53 gene (rAd-p53) (Wills et al.,Human Gene Therapy 5:1079-1088 (1994)) was also tested in this animalmodel of bladder cancer. Briefly, bladder tumors were induced in femaleFisher rates (Charles River) by addition of 0.05% BBN(N-butyl-N-N(4-hydroxybutyl)nitrosamine) in the drinking water for threemonths. rAd-p53 (1×10¹¹ PN/ml) was formulated in 7 mM Big CHAP(CALBIOCHEM®). Under isoflurane anesthesia a catheter (24 G) wasinserted into the bladder for administration. rAd-p53 was instilled intothe bladder for 45 minutes. The animals were then allowed to recoverfrom anesthesia. Twenty-four hours later, animals were sacrificed, andthe bladder was fixed in formalin. After paraffin embedding andsectioning, p53 expression was assayed by immunohistochemistry usingp53ES-kit (Oncogene) using AEC (AEC-kit, Vector Labs) as a substrate.Tissues were counterstained with hematoxylin. FIG. 12 shows p53 geneexpression in the surface area of proliferative epithelium (left panel)and nuclear staining for p53 expression at higher magnification (rightpanel). No staining was detected in tumor tissue from untreated animals.

Example 9 Big CHAP (CALBIOCHEM®) Enhances Transgene Expression in PigUrothelium

To simulate volumes expected for clinical investigation, the 7 mM BigCHAP (CALBIOCHEM®) formulation was tested in a chronically catheterizedadult pig model in collaboration with SPRI Drug Safety and Metabolism.rAd-βgal (1×10¹¹ PN/ml) was formulated in VPBS or 7 mM Big CHAP(CALBIOCHEM®). A volume of 50 ml was injected via the catheter into thebladder of the conscious animals. The instilled material was retainedfor 2 hr. The animals were sacrificed 48 hr later, and a central sectionof the bladder was harvested and stained for β-galactosidase expression.An increase in the intensity of gene expression was observed in the 7 mMBig CHAP (CALBIOCHEM®) treated pig compared to the VPBS treated pig(FIG. 11). Histologic evaluation demonstrated transduction of severalepithelial layers using Big CHAP (CALBIOCHEM®) (left panel), but onlysuperficial transduction with the VPBS buffer (right panel).

Example 10 Gene Transfer into Intestinal Epithelium in Rats

A slightly modification of the method of Sandberg et al. (Human GeneTherapy 5:323-329 (1994)) was used to prepare rat ileal segments forgene transfer studies. Briefly, female Sprague-Dawley rats wereanesthetized with isoflurane. The abdominal cavity was opened and anileal segment rostral from the last Peyer's patch isolated. The segment(about 3 cm) was cautiously cleared from food residues and both sidesclosed with a traumatic vascular clamps. rAd-βgal (1×10¹¹ PN/ml), 0.5 mlvolume, was directly injected into the segment with a 24 G needle andallowed to incubate for 45 minutes. rAd-βgal was formulated in 10 mMtaurodeoxycholic acid (in distilled water, sterile filtered) (Treatmentgroup 1) or VPBS (Treatment Group 2). A third treatment group comprisedanimals treated with 10 mM taurodeoxycholic acid. Thereafter, clampswere removed and a loose silk suture anchored on both ends forrecognition at time of necropsy. The abdominal incision was closed andanimals allowed to recover in their cages. Animals were sacrificed 48 hrlater. The infected segment and a control segment were harvested infixative for whole organ Xgal staining.

The results are shown in FIG. 13. The extent of Xgal blue stainingdemonstrated evidence of transgene expression in the ileal sections.Enhanced gene transfer was evident in the detergent formulation (medialpanel).

Example 11 Effect of Impurities in BIG CHAP on Gene Transfer

1. Introduction

Alternate commercial sources of Big CHAP (BC) were tested for theability to enhance rAd (recombinant adenovirus) mediated gene transferand expression, essentially according to the method described above inExample 8. It was determined that the more “pure” BC—Sigma (98% pure;Sigma Catalog: Biochemicals and Reagents for Life Science Research,1997, page 182, #B 9518) at a concentration of 6 mg/ml did not markedlyimprove rAd mediated gene transfer (FIG. 14, top row). In contrast, theBC (CALBIOCHEM®; CALBIOCHEM® Biochemical & Immunochemical Catalog1996/97, page 43, #200965, 95% pure), did substantially enhance genetransfer and expression at the same concentration (FIG. 14, bottom row).

The BC of CALBIOCHEM® and Sigma were further analyzed by TLC andpurified by column chromatography. Purified BC and isolated impuritieswere tested for their ability to enhance rAd mediated gene transfer andexpression in the bladder epithelium.

As discussed below in more detail, three impurities were isolated fromBC. Two of the impurities demonstrated improvement of rAd mediated genetransfer and expression. In addition to commercial BC, both impuritiesare preferred for rAd formulation buffer to improve local gene delivery.

2. Analysis of Big CHAP by Thin Layer Chromatography

BC (Sigma or CALBIOCHEM®) was dissolved in methanol/water, 3/1, and TLCperformed on Silica gel 60, 0.25 mm (EM Industries); the mobile phaseconsisted of: 1-Butanol/Water/Glacial Acetic Acid, 6/2.5/1.5.Chromatograms were visualized with 0.5 g of thymol in sulfuricacid/ethanol, 5/95, and heat. As shown in FIG. 15, only one distinctband developed from the sample of BC—Sigma (B), while three additionalbands became evident in the sample of BC-CALBIOCHEM® (A).

Impurities of BC (CALBIOCHEM®) were further isolated by columnchromatography and analyzed by thin layer chromatography (Silica Gel60), using a mobile phase of chloroform/methanol/water, 6/5/1. Theresults are depicted in FIG. 16. (Lane 1: BC (CALBIOCHEM®); Lane 2:Impurity I; Lane 3: Impurity II; Lane 4: Mixture of Impurity II and III;Lane 5: Impurity III; Lane 6: BC (CALBIOCHEM®) pure; Lane 7: BC(CALBIOCHEM®).

3. Increasing Concentrations of BC (Sigma) Enhance Gene Transfer

To test impurities of BC for enhancement of gene transfer, rAd-βgal(1×10¹¹ PN/ml) was formulated in increasing concentrations of BC (Sigma)and tested in animals as described above. The results are depicted inFIG. 17. A higher concentration, i.e., 20 mg/ml, of the Sigma BCimproved epithelial gene expression (upper and middle panel). Incomparison, similar gene expression was induced by BC (CALBIOCHEM®) at alower concentration (6 mg/ml, FIG. 17, lower panel).

4. BC Purified by Column Chromatography does not Enhance Gene Transfer

rAd-βgal was formulated in 30 mg/ml of the column chromatographypurified material of both BCs and gene transfer to the bladderepithelium tested as described above. At a concentration of 30 mg/ml,gene transfer and expression was only slightly enhanced in theCALBIOCHEM® sample (FIG. 18, upper panel, right). The purified Sigma BCwas without any effect (FIG. 18, lower panel, left). Purification ofboth BCs (Sigma or CALBIOCHEM®) resulted in decreased gene transfer andexpression.

5. A mixture of Impurity II and Impurity III Enhances Gene Transfer

Three impurities of BC (CALBIOCHEM®) were detected by TLC (FIG. 15) andisolated by column chromatography for gene transfer studies. Impurity Iand a mixture of impurity II and impurity III were diluted in VPBS (0.6mg/ml or 6 mg/ml) to test their efficiency in improving rAd mediatedgene transfer to the bladder epithelium. Impurity I did not lead toincreased β-galactosidase gene expression in the bladder epithelium, butrather caused cystitis (FIG. 19, lower panel, right). In sharp contrast,the mixture of impurity II and III enhanced gene transfer and expressiondose dependently (FIG. 19, lower panel, left). Positive controlformulation (BC, CALBIOCHEM®, upper panel, left), and the negativecontrol formulations (BC-CALBIOCHEM®, column chromatography purified andBC—Sigma) were used at a concentration of 6 mg/ml (upper panel, right).

6. Reconstitution of Impurities into Big CHAP Leads to Enhancement ofGene Transfer

In this experiment, 10 mg/ml of BC (Sigma, FIG. 20 upper middle panel)was reconstituted with Impurity III (upper right panel), impurity II(lower left panel), or synthesized analog of impurity II (lower rightpanel). rAd-βgal, 1×10¹¹ PN/ml, was prepared in the spiked formulationsand administered intravesically as described above. As shown in FIG. 20,improved reporter gene expression (β-galactosidase) was observed in thebladder epithelium of the animals that received rAd dissoluted in the“spiked” BC (Sigma) formulations at a concentration of 10 mg/ml Big CHAP(Sigma).

Example 12 Synthesis of 3-Aminopropyl-3′-N-gluconamidopropyi-amine

1.3′-N-gluconamidopropyl-3″-N-cholamidopropyl-N-cholamide

Glucono-δ-lactone (0.11 mol, 17.8 g) is added in small portions to asolution of 0.11 mol (13.1) g of iminobispropylamine in 400 ml ofrefluxing absolute methanol. After refluxing for 2 hours, the solutionis allowed to cool on ice for 1 hour. The solvent is evaporated todryness.

2. 3-Aminopropyl-3′-N-gluconamidopropyl-amine

Triethylamine (0.2 mol, 28 ml) is added to a solution of 0.2 mol (81.6g) of cholic acid dissolved in 500 ml of dry DMF in a 1-liter flask. Thesolution is cooled to 0° C. in an ice-salt bath, after which 0.2 mol (20g) of isobutylchloroformate is added. The mixture is allowed to stand inthe ice-salt bath for 5 min. after which triethylamine hydrochlorideprecipitate is visible. The reaction yields a mixed anhydrideintermediate.

In a separate 2-liter flask, 0.1 mol (30.9 g) of3′-N-gluconamidopropyl-3″-N-cholamidopropyl-N-cholamide is dissolved in500 ml of DMF by gentle warming to 40-60° C. This solution is cooledrapidly in the ice-salt bath just until clouding occurs, at about 10° C.The mixed anhydride intermediate is filtered into the solution of3′-N-gluconamidopropyl-3″-N-cholamidopropyl-N-cholamide in DMF. Thetriethylamine hydrochloride precipitate is removed by filtration.Thereafter, the solution is stirred with cooling for 24 hours. DMF isremoved by evaporation under vacuum and heat, and the crude mixture issubjected to column chromatography on a silica gel withchloroform/methanol/water, 65/5/1, as the mobile phase. Pure fractionsare collected and the solvent evaporated by vacuum. The reaction yieldsabout 27 g (25%) product.

Mass spectral analysis of the product gave the following peaks: 337.2,394.2, 412.2, 503.8, 682.4, 700.5, 755.1, 801.1, 823.1, 912.3, 1054.8,1074.7, 1090.6, 1112.4, 1119.3.

Example 13 Characterization and Synthesis of Transfection-EnhancingComponents in Big CHAP

As demonstrated in Example 11, impurities present in Big CHAP functionto enhance gene transfer. This Example describes furthercharacterization and synthesis of these compounds.

Calbiochem Big CHAP was fractionated by column chromatography to obtainessentially pure impurities “1”, “2”, and “3” for biological testing aswell as structural analysis. Impurity I was not tested for biologicalactivity because of bladder irritation that was observed in initialexperiments. Because Impurities 2 and 3 were not very soluble in water,they were mixed with 6 mg/ml of Sigma Big CHAP at 0.12 and 1.2 mg/mllevels and were found to enhance gene transfer (Sigma Big CHAP alone at6 mg/ml does not enhance gene transfer).

The structures of Impurities 1, 2, and 3 were determined by MALDI-MS andNMR analysis. FIG. 22 shows the structure, MALDI-MS, and ¹H-NMR spectraof Impurity 1. The structure, MALDI-MS, and ¹H-NMR spectra of Impurity 2are shown in FIG. 23, and those of Impurity 3 are shown in FIG. 24.Comparison of the spectra to those of Big CHAP demonstrate that theimpurities arose from the process used to synthesize Big CHAP, ratherthan as degradants of Big CHAP.

Crude Sigma Big CHAP was found to enhance gene transfer when used at aconcentration of 26 mg/ml. To determine whether trace levels ofimpurities were present in Sigma Big CHAP, 1 mg was applied to a silicagel plate. An impurity comigrating with Impurity 2 in Calbiochem BigCHAP was observed. MALDI-MS and NMR confirmed that this impurity had thesame structure as Impurity 2 in Calbiochem Big CHAP. Several grams ofSigma Big CHAP were fractionated by silica gel flash chromatography andthe fractions containing impurities were consolidated, concentrated, andanalyzed by TLC. Several impurities, including Impurities 2 and 3 wereevident in this trace impurity enriched fraction.

Synthesis of Impurity 2

Impurity 2 was synthesized as follows (see FIG. 25). First, Compound IIIas shown in FIG. 25 was synthesized by dissolving 1.78 g (10 mmol) ofgluconolactone in 200 ml of refluxing methanol and adding 4.2 ml (30mmol) of N-3-aminopropyl)-1,3-propanediamene. Refluxing was continuedfor two hours. The methanol was then evaporated on a rotary evaporatorand the resulting oil was triturated with chloroform until a white solidwas formed. The white solid was filtered, washed with chloroform, anddried by suction to yield 2.1 g of product (impure Compound III).

Compound IV was synthesized by dissolving 0.65 g (1.6 mmol) of cholicacid in 40 ml of N,N-dimethylformamide with heating and stirring. Thesolution was then cooled in an ice bath while stirring was maintained.Triethylamine (0.223 ml (1.6 mmol)) was then added, followed by 0.208 ml(1.6 mmol) of isobutylchloroformate. A white precipitate formed as thestirring was continued for ten minutes, with Compound IV remaining insolution.

To synthesize Impurity 2 (Compound V in FIG. 25), 0.5 g (1.6 mmol) ofCompound III was dissolved in 100 ml dimethylsulfoxide by stirring at55° C. The suspension containing Compound IV was filtered into thissolution and the resulting solution was stirred at room temperatureovernight. Attempted separation of the dimethylsulfoxide from theproduct (using half of the reaction mixture) by addition of water andextraction with methylene chloride or methylene chloride/methanol wasunsuccessful. The other half of the reaction mixture was distilled undervacuum to remove most of the dimethylsulfoxide. The residue was purifiedby silica gel flash chromatography using methanol/chloroform (40/60) asthe eluent. Analysis of the fractions eluting from the column wasconducted by silica gel thin layer chromatography using a mobile phaseconsisting of chloroform/methanol/water (6/5/1) and visualization bycharring after spraying with ethanolic sulfuric acid. The fractionscontaining the purest product were consolidated, evaporated to drynessand triturated with hexane to produce a light tan solid which wasfiltered and washed with hexane. ¹H-NMR and MALDI mass spectrometricanalysis of the product were consistent with the structure shown.

Biological evaluation of this compound was somewhat hampered by its lackof solubility in water. However, even when the compound was not fullydissolved, gene transfer to bladder was enhanced by the incompletelydissolved compound. Formulation of Impurity 2 in Big CHAP, for example,did result in a formulation that is effective for enhancing genetransfer to cells.

Synthesis of Syn3 (Impurity 3 Analog)

Since Impurity 3 is more polar, and hence more water soluble, thanImpurity 2, the synthesis of this compound was attempted. Purified BigCHAP was reacted with the mixed anhydride of cholic acid (formed byreacting cholic acid with isobutylchloroformate). The reaction resultedin poor yield and many products, so an analog of Impurity 3 wassynthesized. This analog, which has a polarity similar to that ofImpurity 3, was termed “Syn3”.

Part 1: Synthesis of Compound III

The synthetic scheme for Syn3 is shown in FIG. 26. The lactone oflactobionic acid (II) was synthesized by dissolving one g (2.8 mmol) oflactobionic acid (I) in 50 ml of methanol, evaporating to dryness on arotary evaporator, and repeating this a process six times. To obtainCompound III, the resulting residue (II) was dissolved in 50 ml ofisopropanol by heating to 50° C. To this solution was added 1.2 ml (8.4mmol) of N-3-aminopropyl)-1,3-propanediamene. The temperature wasincreased to 100° C. and the solution was stirred for three hours. Thesolvent was removed by rotary evaporation and the resulting residue waswashed several times with chloroform to remove excess unreactedN-(3-aminopropyl)-1,3-propanediamene. The remaining residue (III) wasused as is in Part 3 below.

Part 2: Synthesis of Compound IV

Compound IV was synthesized by dissolving 2.28 g of cholic acid (5.6mmol) in N,N-dimethylformamide by heating to 60° C. Triethylamine (0.78ml (5.6 mmol)) was added and the solution was cooled in an ice bath.Isobutyl chloroformate (0.73 ml (5.6 mmol)) was then added and a whiteprecipitate formed as the stirring was continued for ten minutes.

Part 3: Synthesis of Syn3 (Compound V)

Compound III was dissolved in N,N-dimethylformamide, cooled in an icebath, and stirred. The suspension resulting from the synthesis ofCompound IV was filtered into the solution containing Compound III. Theresulting solution was stirred at room temperature for 6 hrs. Thesolvent was removed using high vacuum rotary evaporation and the residuewas dissolved in 100 ml of chloroform/methanol (50/50). Twenty-five mlof this solution was purified by silica gel flash chromatography usingchloroform/methanol (60/40) as the elution solvent. Analysis of thefractions eluting from the column was conducted by silica gel thin layerchromatography using a mobile phase consisting ofchloroform/methanol/water/concentrated ammonium hydroxide (100/80/10/5).The compounds were visualized by charring after spraying with ethanolicsulfuric acid. Fractions containing product were consolidated andrepurified using flash chromatography andchlroform/methanol/water/concentrated ammonium hydroxide (100/80/10/5)as the elution solvent. Fractions containing product were consolidatedand evaporated to a white powder (300 mg of Compound V). ¹H-NMR andMALDI mass spectrometric analysis of the product were consistent withthe structure shown.

Syn3 formed a gel when dissolution was attempted at 10 mg/ml in waterand appeared to form vesicles at 1 mg/ml. However, at 1 mg/ml in 0.1%Tween 80 a clear solution of Syn3 resulted. This formulation was foundto enhance gene transfer. Tween 80 alone, when tested, had no effect ongene transfer.

Purified Big CHAP spiked with Impurities 2 or 3 is an effective enhancerof gene transfer. Synthetic Impurity 2 alone and a synthetic analog ofImpurity 3 (Syn3) alone can enhance gene transfer. Therefore, asynergistic relationship between Big CHAP and the impurities is notrequired for gene transfer enhancement. Big CHAP is highly water solubleand is effective in bringing the impurities and their analogs intosolution, probably as mixed micelles, thus serving as a vehicle for theactive impurities and/or analogs.

While Impurity 2 is effective in enhancing gene transfer, its haslimited solubility in aqueous solutions, although it is useful whenformulated in a suitable solubilizing agent such as Big CHAP. Incontrast to Impurity 2, Syn3 is readily solubilized in, for example, 1mg/ml in 0.1% Tween 80 and other aqueous solutions as described herein.Thus, this compound is particularly useful as a gene transferenhancement agent.

Example 14 Efficacy of Synthetic Impurity 3 Analog (Syn3) for EnhancingGene Transfer to the Bladder

This Example demonstrates that the Syn3 analog of Impurity 3 iseffective in enhancing gene transfer to the bladder.

Methods

1. Dissolution of Sy3

Initial testing of Syn3 indicated that it is not highly soluble ineither buffered saline or dH₂O. However, Syn3 was found to be fairlyeasily dissoluted into the detergent Big CHAP, as well as into thedetergent Tween-80 (although with somewhat more difficulty compared todissolution into Big CHAP). The higher the concentration of the Big CHAPsolution used for dissolution, the greater the amount of Syn3 that couldbe dissoluted. Up to 5 mg/ml of Syn3 was found to dissolute into 15 mMBig CHAP.

For the following studies using Syn3 in Tween-80, a 100 mg/ml solutionof Syn3 was prepared in 10% Tween-80. This stock solution was dilutionin dH₂O (1:100) to give a final concentration of 1 mg/ml Syn3 in 0.1%Tween-80.

Table II summarizes the concentrations of Syn3 that were chosen fortesting in vivo:

TABLE II Concentration of Syn3 in Final Concentration detergentFormulation of Syn3 with rAd 5.0 mg/ml 15 mM Big CHAP 4.5 mg/ml 0.5mg/ml 7.8 mM big CHAP 0.45 mg/ml 0.25 mg/ml 3.9 mM Big CHAP 0.22 mg/ml1.0 mg/ml 0.40% Tween-80 0.90 mg/ml 1.0 mg/ml 0.10 Tween-80 0.45 mg/ml0.50 mg/ml 0.05% Tween-80 0.22 mg/ml

2. In Vivo Testing

The gene transfer activity of Syn3 was tested in vivo by determining thelevel of β-galactosidase expression found following, administration ofadenovirus containing the β-galactosidase gene delivered in one of theabove detergent solutions. In this procedure, female HarlanSprague-Dawley rats were catheterized and administered adenovirusdiluted 1:10 in either Big CHAP or Tween-80 containing Syn3 at one ofthe above concentrations for 45 minutes. Following removal of virus andflushing of the bladder, the animals were allowed to recover. After 48hours the animals were sacrificed, their bladders fixed, and stained forβ-gal expression. Following photographic recording, bladders wereembedded in paraffin for sectioning and histological examination.

Results

1. Gene Transfer Activity of Syn3 in Big CHAP

Syn3 was tested at 0.5 mg/ml in 7.8 MM Big CHAP. At this concentration,it was relatively easily dissoluted, and sterile filterable (0.2 μmAcrodisc syringe filter; Gelman Sciences). Initial experiments utilizedCalbiochem Big CHAP, lot # B19546, while later experiments utilizedSigma Big CHAP lot #37H5023. Neither stock of Big CHAP has experimentsgene transfer activity alone at the concentration employed. As apositive control, Calbiochem lot #679793 was utilized as a formulationfor rAd delivery. This particular lot of Big CHAP was identified ascontaining the active impurities which were identified and from whichSyn3 was modeled. As seen in FIG. 27, Syn3 (I3A) was found to greatlyenhance gene transfer and β-gal expression compared to administration ofthe virus alone in 7.8 mM Big CHAP.

To determine whether lower concentrations of Syn3 might prove asefficacious at enhancing gene transfer as higher concentrations, Syn3was administered at 0.25 mg/ml in 3.9 mM Big CHAP (FIG. 28A). Very highlevels of gene transfer were obtained, but not quite as consistentlyhigh as seen with Syn3 at 0.5 mg/ml (FIG. 28B).

2. Gene Transfer Activity of I3A/Syn3 in Tween-80

Initial testing of I3A in Tween-80 began using I3A at 1 mg/ml in 0.4%Tween-80. However, almost no gene transfer was obtained when thisconcentration of Tween was used (data not shown). Since it washypothesized that the high concentration of Tween-80 might have beensequestering the I3A from partitioning into the membrane and permittingviral penetration, the concentration of Tween-80 was reduced to 0.1%,keeping the concentration of I3A at 1 mg/ml. Two different preparationsof Syn3 were tested for their gene transfer activity at 1 mg/ml in 0.1%Tween-80. At this concentration, very high levels of gene transfer wereseen when using either the first lot (I3A) (FIG. 29A) or the second lotof Syn3 (FIG. 29B). The second lot of Syn3 had also demonstrated veryhigh levels of gene transfer activity at 0.5 mg/ml in 7.8 μM Big CHAP,so all future experiments were carried out using Syn3 instead of I3A.

The gene transfer activity of Syn3 in Big CHAP (0.5 mg/ml in 7.8 MM) wascompared to its activity in Tween-80 (1 mg/ml in 0.1% Tween-80). Bothformulations were found to have approximately equal levels of genetransfer enhancement with perhaps slightly greater transfer seen withthe Syn3 in Big CHAP (FIG. 30A and FIG. 30C respectively). Since theβ-galactosidase assay is not highly quantitative, small differences aredifficult to discern. However, bladders treated with Syn3 at 0.5 mg/mlin 7.8 mM Big CHAP consistently had the highest levels ofβ-galactosidase expression. Syn3 increases gene transfer in eitherdetergent, although it is not as easily dissoluted into the Tween-80.

Since the concentration of Syn3 in Big CHAP was twice that in Tween,Syn3 was next tested for its gene transfer activity at the sameconcentration in these two detergents. At 0.5 mg/ml, Syn3 appeared togive better gene transfer in 7.8 mM Big CHAP (FIG. 31A), than what wasobtained using Syn3 in 0.05% Tween-80. When Syn3 was used at 0.5 mg/mlin 0.05% Tween-80, there appeared to be more regions lackingβ-galactosidase expression, similar to what was observed when theconcentration of Syn3/(I3A) was reduced to 0.25 mg/ml in 3.9 mM Big CHAP(FIG. 28A). This suggests that some of the differences in genetransfer-activity of Syn3 in Big CHAP vs. Tween-80 are probably due inpart to effects from the detergent into which Syn3 was dissolved.

3. Histological Examination of Syn3 Treated Bladders

Bladders from animals treated with Syn3-rAd (β-Gal) were prepared forhistological examination to determine the levels of viral infection, aswell as the degree of viral penetration into the bladder urothelium.Reducing the concentration of Syn3 in either detergent resulted in aconcomitant reduction in β-Gal expression (FIG. 32A-F). Although theβ-galactosidase expression resulting from administration of Syn3 at 0.5mg/ml in 7.8 mM Big CHAP was slightly greater than with Tween-80 (FIG.32A vs. FIG. 32D), it was noted that this concentration of Syn3typically results in a massive recruitment of infiltrates to the bladder(FIG. 32A).

Since the β-galactosidase expression and the level of infiltrates washigher in the bladders in which Syn3 was used at 0.5 mg/ml in Big CHAPthan in the bladders in which Syn3 was at 1 mg/ml in Tween-80, thissuggests that the infiltration was due to the increase in viralpenetration and expression that occurs when rAd is administered in theBig CHAP. In order to discern the contribution that Syn3 may have in therecruitment of infiltrates, sections of bladders that were exposed toSyn3 and virus were compared to those that had been exposed to Syn3alone (FIG. 33A and FIG. 33B, respectively). When Syn3 is administeredalone, a significant amount of infiltration is seen, only slightly lessthan that seen with Syn3 and virus together. Virus administered withoutSyn3 resulted in extremely low levels of infection and infiltrates (FIG.33C), while the negative control (no virus, no Syn3) shows noinfiltration (FIG. 33D).

4. Stability of Syn3 in Solution

Syn3 is very stable when dissoluted into Big CHAP detergent. When Syn3was dissoluted into Big CHAP at either 0.25 mg/ml or 0.5 mg/ml, itretained its gene transfer activity for extended periods (30 days orlonger) even when stored at room temperature. When Syn3 was dissolutedat 100 mg/ml into 10% Tween-80, it was stable for at least one week whenkept at 4° C. However, if left at room temperature at this highconcentration (100 mg/ml), it will solidify within 24 hours. Syn3 thathas been diluted to 1 mg/ml in 0.1% Tween-80 is stable for at least 30days (longest period tested).

Conclusions

The gene transfer activity of Syn3 appears to be extremely high at 0.5mg/ml in 7.8 mM Big CHAP. However, lower concentrations of Syn3 arepreferred (e.g., 0.25 mg/ml in 3.9 mM Big CHAP) due to the possibilityof side effects at higher concentrations. Syn3 has also demonstratedconsistently high levels of gene transfer at 1 mg/ml in 0.1% Tween-80.Based upon the results of these studies, one particularly suitableformulation of Syn3 for use as a gene transfer agent is at 1 mg/ml in0.1% Tween-80.

Example 15 Clinical Formulation of Syn3

This Example provides, for illustrative purposes, one example of aformulation of Syn3 that is suitable for use as a clinical formulationfor delivery of a viral vector. This formulation can also be used forother delivery enhancing compounds; many other formulations such asthose described herein are also suitable for use with Syn3 and othercompounds.

A Syn3 stock solution was prepared by dissolving Syn3 at 100 mg/ml in10% Tween 80. This stock solution was then diluted to a Syn3concentration of 6 mg/ml using an aqueous buffer containing Tris (1.7mg/ml), sodium phosphate (monosodium, dihydrate, 1.7 mg/ml), sucrose (20mg/ml), magnesium chloride (hexahydrate, 0.4 mg/ml), and glycerol (100mg/ml) in water.

This solution was diluted with a solution containing the viral vector toobtain a virus solution that contained 1 mg/ml Syn3 in 0. 1% Tween 80.This solution was effective in enhancing gene transfer.

Example 16 Synthesis of Syn3 Analogs that have Increased Solubility inWater

Syn3 has demonstrated high gene transfer-enhancing activity in vivo, butis relatively insoluble in aqueous solutions, and requires the presenceof detergent for complete dissolution. In addition, Syn3 requiresseveral hours to completely dissolute into 10% Tween-80, furthercomplicating clinical use of this reagent. To resolve thesedifficulties, two analogs of Syn3 were synthesized which have greatersolubility in aqueous solution. By removal of the lactose moiety of Syn3and subsequent methylation or reduction of the resulting amine, twonovel compounds were synthesized which are known as A-Trimethylammoniumchloride (A-tma) and A-Hydrochloride (A-HCl), respectively, where Arepresents the conserved region of Syn3 common to both molecules (seeFIG. 21). These two cationic compounds were further neutralized to theirchloride salt for ease of dissolution.

A-TMA was synthesized as shown in FIG. 35. Briefly, cholic acid (CA)(2.0 g, 5 mmol) in DMF (30 mL, 0° C.) was treated with Et₃N (0.72 mL,5.1 mmol) and then, with care, isobutyl chloroformate (0.67 mL, 5.1mmol). The mixture was stirred for three days and resulted in onecompound. This was readily purified on SiO₂, eluting with DCM/MeOH (6:1to 4:1). After 30 min. at room temperature, a solution of the amine(J-2/55) (522 mg, 2.26 mmol) in DMF (4 mL) was added. The amine wassynthesized according to Han, Y- P and Hang, H- S, Bull. Korean Chem.Soc. (1994) 15: 1025-1027. 1.8 g of the resulting compound, J-2/5C(BOC-A), was obtained, resulting in a 72% yield.

The amine (250 mg, 61/11, 0.27 mmol) in DMF (10 mL) was treated withHungs base (diisopropylethylamine) (200 μL, 1 .15 mmol) and Mel (75 μL,1.2 mmol). TLC showed mainly one compound, plus a few impurities. Thereaction mixture was concentrated under vacuum and applied to a silicacolumn eluted with MeCN/AcOH/H₂O) (4:1:1). The middle fractions werecombined and subjected to ion exchange chromatography on the Na⁺ form ofDowex 50W-X8-200 cation exchange resin, eluted with 1:10.5 M NaCl/MeOH.The purest fractions were desalted on LH-20 lipophilic Sephadex andlyophilized to give the pure trimethyl ammonium chloride (J-2/90(A-tma)). 82 mg of the resulting compound was obtained, resulting in a32% yield.

To obtain A-HCl, BOC-A (1.0 g, 1 mmol) in MeOH (60 mL) was treated witha solution of AcCl in MeOH (2 mL in 20 mL) at 0° C. The reaction wasslowly allowed to attain room temperature. TLC after 3 h showed nostarting material. Following evaporation (with EtOH/toluene), theresidue was applied to the Na⁺ form of a Dowex 50W-X8-200 ion exchangecolumn. However, the product passed straight through. Elution with 0.5 MNaCl did not produce any further material. Flash chromatography on SiO₂was successful (DCM/MeOH/H₂O; 60:35:5), although the product seemed toelute in two bands. NMR showed the late and early fractions to be thesame. 650 mg of the resulting compound, A-HCl, was obtained, resultingin a 65% yield. It was noted that base treatment (-Ome or resin) canchange the TLC behavior of the product.

Example 17 A-tma and A-HCl Enhance Gene Transfer in Vivo

This Example demonstrates that the compounds A-tma and A-HCl have genetransfer activity in vivo.

Methods

1. Preparation of Solutions for Administration:

A concentration of 1 mg/ml was chosen for initial testing of bothcompounds. For determination of the gene transfer activity for each ofthese compounds, the level of β-galactosidase activity obtainedfollowing administration of Syn3 analog/virus/buffer was compared to theactivity when using the virus/buffer alone.

The solution of A-TMA was prepared by dissoluting 10 mg of A-TMA into 10ml Dulbecco's PBS. Glycerol was added to provide a final concentrationof 10 mg/ml. All solutions were sterile filtered prior to use (0.2 μmAcrodisc syringe filter). The virus (BGCG 70AAB) was diluted 1:10 intoeither this A-TMA solution, or into Dulbecco's PBS-glycerol prior toadministration.

Since A-HCl is not completely soluble in saline, and since dissolutioninto dH₂O resulted in a solution whose pH was 4.7, a Tris-bufferedsolution was chosen for dissolution of A-HCl whose composition was asfollows:

Dissolution buffer (Buffer D)

2.8 mM Tris, pH 7.5

1.2 mM NaH₂PO₄

2 mM MgCl₂

0.2% sucrose

10 mg/ml glycerol

Final pH 6.5

Ten mg of A-HCl was dissoluted into this buffer and sterile filteredprior to use (0.2 μm Acrodisc suring filter). The virus (BGCG 70AAB) wasdiluted 1:10 into this solution prior to administration. For comparison,the virus diluted into this buffer without A-HCl was also tested.

2. In Vivo Administration

Female HSD rats were anesthetized using isofluorane. The rats weretrans-uretherally catheterized into the bladder using PE50 tubinglubricated with K-Y jelly. A tie-off was installed on the externalurethra to prevent back leakage. Urine, if any, was removed and thebladder was flushed with 0.5 ml PBS and emptied. rAd was diluted to thedesired concentration (1:10) and instilled for 45 minutes. The dosingmaterial was removed, noting the return volume. The bladder was flushedwith 0.5 ml PBS and emptied. The tie-off and catheter were removed, andthe animals were allowed to recover in cages.

After 48 hours, the animals were sacrificed and their bladders inflationfixed with 0.5 ml fixative for 1 hour. The bladders were then rinsedovernight and whole organ X-gal staining was conducted.

Results

The two compounds both gave enhanced gene transfer activity compared tocontrols. The levels of gene transfer activity are summarized in TableIII. Relative levels of gene transfer activity are shown; highest levelsof gene transfer activity are indicated by ‘++++’, and low levels areindicated by ‘+’ (no transfer activity indicated by 0).

TABLE III Assessment of gene transfer activity in animals usingwater-soluble Syn3 analogs Animal # Solution Composition Gene TransferActivity #297 PBS/1% Glycerol 0 #298 1 mg/ml A-TMA ++ in PBS/1% Glycerol#988 1 mg/ml A-TMA ++ in PBS/1% Glycerol #989 1 mg/ml A-TMA ++ in PBS/1%Glycerol #384 Buffer D 0 #385 1 mg/ml A-HCl ++ in Buffer D #386 1 mg/mlA-HCl ++ in Buffer D #387 1 mg/ml A-HCl ++ in Buffer D

Conclusions

The two compounds A-tma and A-HCl both demonstrated gene transferactivity significantly above those levels obtained by controls. Althoughthese levels are lower than those obtained using Syn3 in Tween-80, theydo indicate that gene transfer enhancement is possible using an aqueousbased Transfection Enhancing Agent such as A-tma and A-HCl. The compoundA-SC, in which the lactose moiety of Syn3 is replaced with a succinicanhydride moiety was not effective as a gene transfer enhancingcompound. This compound gave gene transfer activity at levels equal tocontrols (data not shown). Table IV summarizes the gene transfer resultsusing these compounds compared to the gene transfer activity of Syn3 at1 mg/ml.

TABLE IV Summary of gene transfer activity of water-soluble Syn3 analogsCompound Concentration Gene transfer activity A-TMA 1 mg/ml ++ A-HCl 1mg/ml ++ A-SC 1 mg/ml 0/+ Syn3 1 mg/ml ++++

Since both compounds have been found to have much greater solubility indH₂O (up to 5 mg/ml), it is likely that increasing the concentration ofthese analogs will result in even greater gene transfer activity invivo.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference in their entirety asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference.

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above, without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above, are, therefore to be considered asillustrative and not restrictive. The scope of the present invention isas set forth in the appended claims rather than being limited to theexamples contained in the foregoing description.

What is claimed is:
 1. A compound of Formula III:


2. A compound of Formula IV:


3. A compound of Formula V:


4. A delivery enhancing compound of Formula II:

wherein: X₁ and X₂ are selected from the group consisting of

and X₃ is a saccharide group.
 5. The compound according to claim 4,wherein X₁ and X₂ are both

and X₃ is a glucose group.